Studies of function and transcriptional regulation of auxin-induced genes

生长素诱导基因的功能和转录调控研究

• 批准号: 06454011
• 负责人: TAKAHASHI Yohsuke
• 金额: $ 4.8万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06454011/
• 关键词: auxin; transcriptional regulation; transgenic plants; WD-40; two-hybrid; GTP結合タンパク質

We have studied about functions and modulation of expression of auxin-regulated genes, arcA,parB and parC to gain insight into the action mechanism of auxins at the molecular level. The initiation of expression of arcA,isolated from tobacco BY-2 cells, by the application of auxin precedes the induction of cell division. Sequence analysis revealed that arcA belongs to an expanding gene family, including the genes for beta subunits of G proteins. These proteins all have a series of internal repeats of 40 amino acids called the WD-40 repeat. Members of the WD-40 repeat family are involved in various cellular functions. Most known WD-40 proteins form multiprotein complexes, sometimes interacting with other proteins through the WD-40 repeat region. So we isolated several cDNA clones of arcA-interacting proteins using two-hybrid screen in yeasts and examined their direct binding capabilities with arcA products by the in vitro system. One of these cDNA clones had the extensive homology to a beta subunit of voltage-dependent K^+ channel well characterized in mammalian cells. The antibodies against arcA products revealed that arcA products accumulated at the maximum level 2 days after the subculture of BY-2, at which time the cells were most actively dividing.We investigated auxin-responsive cis elements of parA,parB and parC from tobacco mesophyll protoplasts in transgenic tobaccos using GUS as a reporter gene. Detailed analysis including point mutation experiments showed that the interaction of separately located sequences was required for the auxin-responsiveness. Furthermore, different nuclear proteins bound to each of auxin-responsive elements of these genes. Although parA,parB and parC are all isolated from tobacco mesophyll protoplasts, auxin mediated activation of transcription depends on different cis and trans factors. These results suggest that various physiological effects of auxins are underlain by the composite molecular mechanisms of trascriptional regulation.

我们对生长素调控基因ArcA、PARB和ParC的功能和表达调控进行了研究，以期在分子水平上深入了解生长素的作用机制。从烟草BY-2细胞中分离出来的ArcA基因在细胞分裂诱导之前被生长素启动表达。序列分析表明，ArcA属于一个扩展基因家族，包括G蛋白的β亚基基因。这些蛋白质都有一系列由40个氨基酸组成的内部重复序列，称为WD-40重复序列。WD-40重复序列家族的成员参与多种细胞功能。大多数已知的WD-40蛋白质形成多蛋白复合体，有时通过WD-40重复区域与其他蛋白质相互作用。因此，我们在酵母中用双杂交筛选法分离了几个与ArcA相互作用蛋白的cDNA克隆，并通过体外系统检测了它们与ArcA产物的直接结合能力。其中一个克隆与哺乳动物细胞中电压依赖性K^+通道的一个β亚基具有广泛的同源性。对ArcA产物的抗体检测表明，在By-2继代培养2天后，Arca产物积累达到最高水平，此时细胞红利最活跃。我们以GUS基因为报告基因，从烟草叶肉原生质体中研究了生长素应答顺式元件PARB、PARB和PARC。包括点突变实验在内的详细分析表明，生长素反应需要独立定位序列的相互作用。此外，不同的核蛋白与这些基因的每一个生长素反应元件结合。虽然ParA、PARB和ParC都是从烟草叶肉原生质体中分离出来的，但生长素介导的转录激活依赖于不同的顺式和反式因子。这些结果表明，生长素的各种生理作用是由转录调控的复合分子机制所支持的。

项目成果

Nagata,T.et al.: "Genes involved in the dedifferentiation of plant cells." Internatl.J.Develop.Biol.38. 321-327 (1994)

Nagata,T.et al.：“参与植物细胞去分化的基因。”

Ishida, S., Takahashi, Y.and Nagata, T.: "The mode of expression and promoter analysis of auxin-regulated arcA gene." Plant Cell Physiol.37. 439-448 (1996)

Ishida, S.、Takahashi, Y. 和 Nagata, T.：“生长素调节的 arcA 基因的表达模式和启动子分析。”

Nagata, T., Ishida, S., Hasezawa, S.and Takahashi Y.: "Genes involved in the dedifferentiation of plant cells." Internatl. J.Develop. Biol.38. 321-327 (1994)

Nagata, T.、Ishida, S.、Hasezawa, S.和 Takahashi Y.：“参与植物细胞去分化的基因。”

Takahashi, Y., Hasezawa, S., Kusaba, M.and Nagata, T.: "Expression of the auxin-regulated ParA gene in transgenic tobacco and nuclear localization of its gene products." Planta. 196. 111-117 (1995)

Takahashi, Y.、Hasezawa, S.、Kusaba, M.和 Nagata, T.：“生长素调节的 ParA 基因在转基因烟草中的表达及其基因产物的核定位。”

Takahashi,Y.et al.: "Auxin-regulated genes." Plant Cell Physiol.36. 383-390 (1995)

Takahashi,Y.et al.：“生长素调节基因。”